1. HIV Immunotherapy
HIV is the focus of intense studies as it is the causative agent for acquired immunodeficiency syndrome (AIDS). Immunotherapeutic methods are one of several approaches to prevention, cure or remediation of HIV infection and HIV-induced diseases. Specifically, the use of neutralizing antibodies in passive immunotherapies is of central importance to the present invention.
Passive immunization of HIV-1 infected humans using human sera containing polyclonal antibodies immunoreactive with HIV has been reported. See for example, Jackson et al., Lancet, September 17:647-652, (1988); Karpas et al., Proc. Natl. Acad. Sci., USA, 87:7613-7616 (1990).
Numerous groups have reported the preparation of human monoclonal antibodies that neutralize HIV isolates in vitro. The described antibodies typically have immunospecificities for epitopes on the HIV glycoprotein gp160 or the related glycoproteins gp120 or gp41. See, for example Karwowska et al., Aids Research and Human Retroviruses, 8:1099-1106 (1992); Takeda et al., J. Clin. Invest., 89:1952-1957 (1992); Tilley et al., Aids Research and Human Retroviruses, 8:461-467 (1992); Laman et al., J. Virol., 66:1823-1831 (1992); Thali et al., J. Virol., 65:6188-6193 (1991); Ho et al., Proc. Natl. Acad. Sci., USA, 88:8949-8952 (1991); D'Souza et al., AIDS, 5:1061-1070 (1991); Tilley et al., Res. Virol., 142:247-259 (1991); Broliden et al., Immunol., 73:371-376 (1991); Matour et al., J. Immunol., 146:4325-4332 (1991); and Gorny et al., Proc. Natl. Acad. Sci., USA, 88:3238-3242 (1991). For a current review of pathogenesis of HIV infection and therapeutic modalities including the use of passive immunity with anti-HIV antibodies, see Levy, Microbiol. Rev., 57:183-289 (1993).
To date, none of the reported human monoclonal antibodies have been shown to be effective in passive immunization therapies. Further, as monoclonal antibodies, they all each react with an individual epitope on the HIV envelope surface glycoproteins, gp120 or gp160, or against the V3 loop of gp120 or against the envelope transmembrane glycoprotein, gp41. The epitope against which an effective neutralizing antibody immunoreacts has not been identified.
There continues to be a need to develop human monoclonal antibody preparations with significant HIV neutralization activity. In addition, there is a need for monoclonal antibodies immunoreactive with additional and diverse neutralizing epitopes on HIV gp120. Additional (new) epitope specificities are required because, upon passive immunization, the administered patient can produce an immune response against the administered antibody, thereby inactivating the particular therapeutic antibody.
Furthermore, the well documented ability of HIV to mutate its envelope glycoprotein structure and thereby alter its reactivity with the immune system of an infected host produces variant "field isolates" which compromise the utility of individual antibody preparations immunoreactive with an individual laboratory strain of HIV. Existing antibody preparations tend to be less potent against primary field isolates of HIV than against laboratory strains. Moore et al., Perspectives in Drug Discovery and Design, 1:235-250 (1993). In addition, no reported human monoclonal antibody has been shown to be effective at neutralizing multiple strains of HIV. Therefore, there also continues to be a need for a human monoclonal antibody with the ability to neutralize multiple different strains of HIV.
2. Human Monoclonal Antibodies Produced From Combinatorial Phagemid Libraries
The use of filamentous phage display vectors, referred to as phagemids, has been repeatedly shown to allow the efficient preparation of large libraries of monoclonal antibodies having diverse and novel immunospecificities. The technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene during the assembly stage of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries. Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991). Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor (Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366, 1991) and the cpIII membrane anchor. Barbas et al., Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991).
The diversity of a filamentous phage-based combinatorial antibody library can be increased by shuffling of the heavy and light chain genes (Kang et al., Proc. Natl. Acad. Sci., USA, 88:11120-11123, 1991), by altering the CDR3 regions of the cloned heavy chain genes of the library (Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461, 1992), and by introducing random mutations into the library by error-prone polymerase chain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992).
Filamentous phage display vectors have also been utilized to produce human monoclonal antibodies immunoreactive with hepatitis B virus (HBV) or HIV antigens. See, for example Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); and Burton et al., Proc. Natl. Acad. Sci., USA, 88:10134-10137 (1991), respectively. Human monoclonal antibodies displayed on the surface of bacteriophage through the use of phage vectors, where the antibodies are specific for HIV-1 antigens, gp120 and gp41, have been generated through screening of combinatorial libraries. The resultant antibodies have been shown to be immunoreactive with HIV and to neutralize HIV. See, Barbas et al., J. Mol. Biol., 230:812-823 (1993); Williamson et al., Proc. Natl. Acad. Sci., USA, 90:4141-4145 (1993); Burton et al., Chem. Immunol., 56:112-126 (1993); and Barbas et al., Proc. Natl. Acad. Sci., USA, 89:9339-9343 (1992).
While the above-described phage display-derived anti-HIV antibodies have been shown to neutralize HIV infection, the screened antibodies are representative of the immune repertoire of an immunized or infected host. However, the heavy and light chain pairings isolated for their affinity for an antigen in vitro are not necessarily paired in vivo. Although the phage display system allows for unique pairing of heavy and light chains, in many cases affinity selection restores the approximate pairings. Burton et al., Nature, 359:782-783 (1992). While such immunized sources or immune priming by natural infection provides useful antibody libraries for some antigens, it is not always possible to acquire such libraries.
Although anti-HIV-1 neutralizing antibodies have been obtained through screening of phage libraries prepared from HIV-1 positive donors, the resultant antibodies are limited in specificity and affinity by the heavy and light chain amino acid residue sequences.
The diversity of a filamentous phage-based combinatorial antibody library, however, can be increased by shuffling of the heavy and light chain genes obtained from an initial screen of a library (Kang et al., Proc. Natl. Acad. Sci. USA, 88:11120-11123, 1991). Another approach is to introduce random mutations into the heavy and light chain genes by error-prone polymerase chain reactions (PCR). Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580, 1992). Mutagenesis of proteins has been utilized to alter the function, and in some cases the binding specificity, of a protein. Typically, the mutagenesis is site-directed, and therefore laborious depending on the systematic choice of mutation to induce in the protein. See, for example Corey et al., J. Amer. Chem. Soc., 114:1784-1790 (1992), in which rat trypsins were modified by site-directed mutagenesis. More recently, Riechmann et al., Biochem., 32:8848-8855 (1993), described the use of site-directed mutagenesis and phage display techniques prior to screening the randomized library to increase the affinity of a single-chain Fab fragment specific for the hapten 2-phenyloxazol-5-one.
A preferred approach, in order to more extensively sample the potential of antibody structure and function, is the preparation of semisynthetic antibodies in the context of phage display. In these molecules, one or more of the complementarity determining regions (CDR) of the cloned heavy or light chain genes obtained from screening of the library are altered resulting in new variable domain amino acid residue sequences. Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992). Unlike antibodies cloned from a particular donor, semisynthetic antibodies can have CDR of any size with any sequence, thereby increasing the potential to obtain antibodies having new specificities and affinities.